Current work in the cell cycle regulation project is aimed at determining the structural basis of mitotic-specific ets-1 hyperphosphorylation and investigating its possible cellular function. This hyperphosphorylated isoform of ets-1 was previously discovered in cells synchronized in mitosis by a variety of methods and was also detected in unsynchronized cells exposed to the phosphatase inhibitor, okadaic acid. Peptide mapping studies indicate that the hyperphosphorylation site lies within exon 7 of ets-1 in a region which contains seven serines arranged near a Ca++/calmodulin kinase consensus site. Ets-1 proteins mutated in each of these serines are being studied to determine effects on in vitro phosphorylation by this and related kinases, and will be utilized in transfection experiments to investigate in vivo effects. In another ongoing project, P19 cells are used as a model system for studying the role of ets proteins in astrocyte differentiation. These multipotential cells can be induced by retinoic acid to differentiate into neurons and astrocytes over a 14-day period of culture. Ets-1 mRNA is induced early in this process and expression is maintained continuously. Conditions which favor neuronal vs. astrocytic development result in disappearance of the ets-1 RNA. Ets-2 mRNA showed a similar pattern of expression, except that treatment with DMSO, which induces muscle cell characteristics in these cells, also induced ets-2, but not ets-1 mRNA. We are now testing this correlation further by investigating whether direct transfection of ets-1 expression in P19 cells can promote astrocyte differentiation independent of retinoic acid exposure.